Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe complications in immune-suppressed or -compromised individuals. Particularly affected among the adult population are AIDS patients as well as patients undergoing immune-suppressive therapy after organ transplantation. In addition, HCMV can cross the placenta and infect the fetus in utero leading to congenital defects. HCMV gene expression is characterized by three phases, immediate early (IE), early and late. After the IE phase of gene expression, numerous early genes are expressed in preparation for viral DNA replication. The early genes are a complex class of genes that 1) differ in their kinetic appearance within the early phase and 2) are expressed at varying abundance at early and late times. Late gene expression is characterized by a continuation of the expression of early genes that are differentially regulated at late times after infection and true late genes that are expressed only after the onset of viral DNA replication. The regulation of viral early and late promoters is being actively examined by several research groups. Sequences that regulate these promoters in cis have been identified. However, studies to date have been performed in transient assays which exclude the influence of the viral genome in regulating early and late gene expression. In these studies, we propose to assess three previously characterized HCMV promoters, pol, pp65 and pp28, by analyzing those cis-acting sequences responsible for regulating gene expression in the context of the viral genome. This will be accomplished by way of the following specific aims: 1) Generation of recombinant virus expressing promoter-reporter gene constructs. 2) Analysis of the kinetics of reporter gene expression at early and late times after infection and comparison to the endogenous gene. 3) Assessment of cis-acting sequences that regulate early promoters. 4) Comparative assessment of the early-late transition by analyzing early and late promoters in the presence and absence of viral DNA replication.